NO330512B1 - Use of 4-H-1-benzopyran-4-one derivatives for the preparation of a pharmaceutical preparation for the inhibition of smooth muscle cell proliferation - Google Patents
Use of 4-H-1-benzopyran-4-one derivatives for the preparation of a pharmaceutical preparation for the inhibition of smooth muscle cell proliferation Download PDFInfo
- Publication number
- NO330512B1 NO330512B1 NO20013335A NO20013335A NO330512B1 NO 330512 B1 NO330512 B1 NO 330512B1 NO 20013335 A NO20013335 A NO 20013335A NO 20013335 A NO20013335 A NO 20013335A NO 330512 B1 NO330512 B1 NO 330512B1
- Authority
- NO
- Norway
- Prior art keywords
- flavopiridol
- alkyl
- proliferation
- cell
- smooth muscle
- Prior art date
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Description
O ppfinnelsens område The field of the invention
Foreliggende oppfinnelse angår anvendelse av 4-H-l-benzopyran-4-one-derivater for fremstilling av et farmasøytisk preparat for inhibering av glatt muskelcelle (SMC) proliferering. The present invention relates to the use of 4-H-1-benzopyran-4-one derivatives for the production of a pharmaceutical preparation for inhibiting smooth muscle cell (SMC) proliferation.
O ppfinnelsens bakgrunn The background of the invention
Den cellulære respons på vaskulær skade - cellulær dysfunksjon, aktivering, dedifferensiering, proliferering og migrering - kulminerer i kliniske hendelser slik som restenoser, som oppstår etter ballongangioplasti og plassering av stenter for behandling av human aterosklerotisk sykdom \ Glatt muskelcelle (SMC)-proliferering er et vanlig og muligens ensartet trekk ved modeller for vaskulær skade, og SMC er hovedcellekomponenten ved neointimale lesjoner<2>'<3>. Ny interesse for å inhibere SMC-proliferering har fulgt økningen i anvendelse av stenter ved behandling av koronar sykdom, siden restenoser ved stenter nesten utelukkende er avhengig av neointimal dannelse og SMC-hyperplasi<4>. Det er beregnet at så mange som 100 000 pasienter med stentrestenoser krevde behandling bare i 1997<5>; derfor ville en lett administrerbar effektiv inhibitor av SMC-hyperplasi ha store kliniske og økonomiske følger<6>. The cellular response to vascular injury - cellular dysfunction, activation, dedifferentiation, proliferation and migration - culminates in clinical events such as restenoses, which occur after balloon angioplasty and placement of stents for the treatment of human atherosclerotic disease \ Smooth muscle cell (SMC) proliferation is a common and possibly uniform feature of models of vascular injury, and SMCs are the major cellular component of neointimal lesions<2>'<3>. New interest in inhibiting SMC proliferation has followed the increase in the use of stents in the treatment of coronary disease, since stent restenosis is almost exclusively dependent on neointimal formation and SMC hyperplasia<4>. It is estimated that as many as 100,000 patients with stent restenosis required treatment in 1997 alone<5>; therefore, an easily administered effective inhibitor of SMC hyperplasia would have major clinical and economic implications<6>.
Forsøk på å inhibere SMC-proliferering i modeller for vaskulær skade, enten ved å modulere cellemediatorer av den proliferative respons, eller ved direkte å interferere med cellesyklusmaskineriet, har medført viktig innsikt i neointimal dannelse. Cellesyklusprogresjon er en tett kontrollert hendelse regulert positivt ved syklinavhengige kinaser (Cdk) og deres syklinregulerende subenheter<7>, og negativt ved Cdk-inhibitorer og tumorsupressorgener slik som retinoblastomprotein ((Rb) og p53<8>. Adenovirus-mediert overekspresjon av endogene Cdk-inhibitorer p21 og ^ 27^ eller av en konstitutiv aktiv form av Rb blokkerer neointimal dannelse i en rotte carotis skademodell9-1 1; tilsvarende inhiberer også inhibisjon av den aktive transkripsjonsfaktoren E2F ved kompetitiv overekspresjon av liknende DNA-bindingsseter SMC-proliferasjon og neointimal dannelse<12>. Slike studier støtter den generelle hypotese at cellesyklusinhibisjon er et attraktivt mål for intervensjon i vaskulær lesjonsdannelse. Attempts to inhibit SMC proliferation in models of vascular injury, either by modulating cell mediators of the proliferative response, or by directly interfering with the cell cycle machinery, have led to important insights into neointimal formation. Cell cycle progression is a tightly controlled event regulated positively by cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits<7>, and negatively by Cdk inhibitors and tumor suppressor genes such as retinoblastoma protein ((Rb) and p53<8>. Adenovirus-mediated overexpression of endogenous Cdks -inhibitors p21 and ^ 27^ or of a constitutively active form of Rb block neointimal formation in a rat carotid injury model9-1 1; similarly, inhibition of the active transcription factor E2F by competitive overexpression of similar DNA binding sites also inhibits SMC proliferation and neointimal formation <12> Such studies support the general hypothesis that cell cycle inhibition is an attractive target for intervention in vascular lesion formation.
Mens intervensjoner har bistått analysen av mekanismene som regulerer neointimal dannelse, lider de av den utilstrekkelighet av for tiden ikke å være klinisk egnet for behandling av vaskulær sykdom hos menneske. En vannløselig, lavmolekylær forbindelse med spesielle cellesyklusregulerende effekter, i særdeleshet en med oral aktivitet, ville ha bred anvendelse både eksperimentelt og eventuelt klinisk. Det nylig identifiserte flavon, flavopiridol, er en Cdk-inhibitor som sterkt blokkerer aktiviteten til Cdk2, Cdc2og CdLt<13>"<16>.1 motsetning til andre farmakologiske inhibitorer av Cdk utmerker flavopiridol seg med sin kinasespesifisitet, sin orale tilgjengelighet og sin styrke ved å være effektiv i nanomolare konsentrasjoner<16>. Disse unike trekk resulterer i en fordelaktig bivirkningsprofil som har ført til utprøving av flavopiridol i fase 1 kliniske studier for behandling av refraktære neoplasmaer 17. Gitt disse egenskaper er flavopiridols evne til å inhibere SMC-proliferering in vitro og etter ballongskade på carotisarterien hos rotte nå undersøkt. Det er funnet at flavopiridol er en potent og selektiv inhibitor av cellesyklusprogresjon og at den arresterer SMC-prolifereringen både in vivo og in vitro; videre blokkeres den neointimale dannelse effektivt ved orale doser av flavopiridol lavere enn de som er kjent for å ha toksiske effekter hos menneske. While interventions have aided the analysis of the mechanisms that regulate neointimal formation, they suffer from the inadequacy of currently not being clinically suitable for the treatment of human vascular disease. A water-soluble, low molecular weight compound with special cell cycle regulatory effects, particularly one with oral activity, would have wide application both experimentally and possibly clinically. The recently identified flavone, flavopiridol, is a Cdk inhibitor that strongly blocks the activity of Cdk2, Cdc2, and CdLt<13>"<16>.1 unlike other pharmacological inhibitors of Cdk, flavopiridol excels in its kinase specificity, its oral availability, and its potency by being effective at nanomolar concentrations<16>.These unique features result in an advantageous side effect profile that has led to the trial of flavopiridol in phase 1 clinical trials for the treatment of refractory neoplasms 17. Given these properties, the ability of flavopiridol to inhibit SMC proliferation in vitro and after balloon injury of the rat carotid artery now investigated. It is found that flavopiridol is a potent and selective inhibitor of cell cycle progression and that it arrests SMC proliferation both in vivo and in vitro; moreover, neointimal formation is effectively blocked by oral doses of flavopiridol lower than those known to have toxic effects in humans.
Det er nå overraskende blitt funnet at 4-H-l-benzopyran-4-one-derivater er egnede SMC-prolifereringsinhibitorer. Det er kjent at 4-H-l-benzopyran-4-one-derivater er egnede for å kontrollere tumorer. Imidlertid er det overraskende at 4-H-l-benzopyran-4-one-derivater som anvendes ved foreliggende oppfinnelse effektivt virker som en SMC-prolifereringsinhibitor ved dosenivåer lavere enn dosenivåene som anvendes ved kontroll av tumorvekst. It has now surprisingly been found that 4-H-1-benzopyran-4-one derivatives are suitable SMC proliferation inhibitors. It is known that 4-H-1-benzopyran-4-one derivatives are suitable for controlling tumors. However, it is surprising that 4-H-1-benzopyran-4-one derivatives used in the present invention effectively act as an SMC proliferation inhibitor at dose levels lower than the dose levels used in controlling tumor growth.
Følgelig er en hensikt ifølge oppfinnelsen å anvende 4-H-l-benzopyran-4-one-derivater som inhibitorer på glatt muskel proliferering. Consequently, an aim according to the invention is to use 4-H-1-benzopyran-4-one derivatives as inhibitors of smooth muscle proliferation.
Figur 1. Effekt av flavopiridol på HASMC DNA- syntese. A. Hvilende HASMC ble behandlet i fravær (-) eller nærvær (+) av bFGF (10 ng/ml) og med de antydede konsentrasjoner av flavopiridol (nmol/1) i 24 timer. BrdU-inkorporering som et mål på proliferering ble bestemt ved en ELISA-basert analyse og uttrykt som prosent inkorporering i fravær av bFGF-behandling.<*>p < 0,05 i forhold til ubehandlede celler. Figure 1. Effect of flavopiridol on HASMC DNA synthesis. A. Resting HASMC were treated in the absence (−) or presence (+) of bFGF (10 ng/ml) and with the indicated concentrations of flavopiridol (nmol/1) for 24 h. BrdU incorporation as a measure of proliferation was determined by an ELISA-based assay and expressed as percent incorporation in the absence of bFGF treatment.<*>p < 0.05 relative to untreated cells.
■ fp < 0,05 i forhold til behandling med bFGF i fravær av flavopiridol. B. HASMC ble behandlet med bFGF (10 ng/ml), trombin (2U/ml), eller vehikkel i nærvær eller fravær av flavopiridol (75nmol/l) og BrdU-inkorporering ble målt. * p < 0,05 i forhold til ubehandlede celler. ** p< 0,05 i forhold til behandling med bFGF alene, fø < 0,05 i forhold til behandling med trombin alene. ■ fp < 0.05 compared to treatment with bFGF in the absence of flavopiridol. B. HASMC were treated with bFGF (10 ng/ml), thrombin (2U/ml), or vehicle in the presence or absence of flavopiridol (75 nmol/l) and BrdU incorporation was measured. * p < 0.05 compared to untreated cells. ** p< 0.05 in relation to treatment with bFGF alone, f< 0.05 in relation to treatment with thrombin alone.
Figur 2. Effekt av flavopiridol på HASMC- proliferering. Hvilende HASMC ble behandlet med bFGF (10 ng/ml) alene (□), bFGF og flavopiridol (75nmol/l) (□), eller vehikkel (□) i den antydede tid og celleantall etter behandling ble bestemt. Resultater er uttrykt som celleantall pr. brønn (x IO<3>). Figure 2. Effect of flavopiridol on HASMC proliferation. Resting HASMC were treated with bFGF (10 ng/ml) alone (□), bFGF and flavopiridol (75 nmol/l) (□), or vehicle (□) for the indicated time and cell numbers after treatment were determined. Results are expressed as cell count per well (x IO<3>).
Figur 3. Effekt av flavopiridol på cyklin- avhengig kinaseaktivitet i HASMC. Hvilende HASMC ble behandlet med bFGF (10 ng/ml), trombin (2 U/ml) eller vehikkel i nærvær eller fravær av flavopiridol (75 nmol/1), og fosforylering av histon Hl ble kvantifisert som et mål på Cdk-aktivitet og uttrykt som en prosent av Cdk-aktivitet i fravær av bFGF-behandling. * p < 0,05 i forhold til ubehandlede celler. ** p < 0,05 i forhold til behandling med bFGF alene, fø < 0,05 i forhold til behandling med trombin alene. Figur 4. Regulering av cellesyklusrelaterte proteiner av flavopiridol. Hvilende HASMC ble behandlet i nærvær (+) eller fravær (-) av bFGF (10 ng/ml), trombin (2 U/ml) og/eller flavopiridol (75nmol/l) i 24 timer. Immunblått av cellelysater ble utført med spesifikke antistoffer som gjenkjenner cyklin Di ( øvre del), PCNA ( midtre del) og fosforylert ( pRb) og hyperfosforylert ( ppRb) Rb ( nedre del). Figur 5. Effekter av flavopiridol på MAP kinaseaktivitet i HASMC. Hvilende HASMC ble behandlet i nærvær (+) eller fravær (-) av bFGF (10 ng/ml), trombin (2 U/ml), PD98059 (30 nmol/1) og/eller flavopiridol (75 nmol/1) i 30 minutter. Nivåer av fosforylert Erkl (pErkl) og Erk2 (pErk2) ble målt ved immunblotting med et fosforylerings-spesifikt antistoff som gjenkjenner begge proteiner (øvre del). MAP-kinase aktiviteten ble målt med en "in-gel" kinaseanalyse, som anvender basisk myelinprotein som et substrat (nedre del). Figur 6. Levedyktigheten til HASMC etter behandling medflavopiridol. Hvilende HASMC ble behandlet med flavopiridol (75nmol/l), TNF-a (50 ng/ml), eller vehikkel for tidsperiodene antydet. Cellelevedyktighet ble vurdert ved eksklusjon av trypanblått. Resultatene uttrykkes som prosent av levedyktige celler i forhold til det totale celleantall. Figur 7. Inhibisjon av neointimal dannelse i rottecarotisarterie ved flavopiridol etter ballongskade. Neointima/media-forholdene ble målt i histologiske snitt av rotte carotisarterier behandlet med eller uten flavopiridol (5 mg/kg) i 5 dager etter skade. Arteriene ble undersøkt 7 ( n = 12) og 14 ( n = 12) dager etter skade. Prosentandelen av PCNA-positive kjerner (± SEM, uttrykt som en prosent av antall talte kjerner) i neointima til arteriene fra hvert tidspunkt og behandlingstype er også gitt. * p < 0,05 sammenliknet med behandling med vehikkel. Figur 8. Histologiske snitt fra rotte carotis arterier. Snittene er fra arteriene 7 ( A og B) og 14 (C og D) dager etter skade. Arteriene vist i A og C er fra rotter behandlet med flavopiridol (5 mg/kg) ved sondeforing; arteriene i B og D er fra rotter behandlet med bare vehikkel. Opprinnelig forstørrelse x 100. Figur 9. Cdk2- ekspresjon etter ballongskade i rotte carotis arterier. Snittene er fira arterier 7 ( A og B) og 14 (C og D) dager etter skade. Arteriene vist i A og C er fira rotter behandlet med flavopiridol (5 mg/kg) ved sondeforing; arteriene i B og D er fra rotter behandlet med bare vehikkel. Cdk2-positive kjerner, lokalisert hovedsakelig i neointima, farges med vektorblått ifølge den alkaliske fosfatasemetoden. Opprinnelig forstørrelse x 100. Figure 3. Effect of flavopiridol on cyclin-dependent kinase activity in HASMC. Resting HASMC were treated with bFGF (10 ng/ml), thrombin (2 U/ml) or vehicle in the presence or absence of flavopiridol (75 nmol/l), and phosphorylation of histone H1 was quantified as a measure of Cdk activity and expressed as a percentage of Cdk activity in the absence of bFGF treatment. * p < 0.05 compared to untreated cells. ** p < 0.05 in relation to treatment with bFGF alone, f < 0.05 in relation to treatment with thrombin alone. Figure 4. Regulation of cell cycle-related proteins by flavopiridol. Resting HASMC were treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml) and/or flavopiridol (75 nmol/l) for 24 hours. Immunoblotting of cell lysates was performed with specific antibodies that recognize cyclin Di (upper part), PCNA (middle part) and phosphorylated (pRb) and hyperphosphorylated (ppRb) Rb (lower part). Figure 5. Effects of flavopiridol on MAP kinase activity in HASMC. Resting HASMC were treated in the presence (+) or absence (-) of bFGF (10 ng/ml), thrombin (2 U/ml), PD98059 (30 nmol/1) and/or flavopiridol (75 nmol/1) for 30 minutes. Levels of phosphorylated Erk1 (pErk1) and Erk2 (pErk2) were measured by immunoblotting with a phosphorylation-specific antibody that recognizes both proteins (upper part). MAP-kinase activity was measured with an "in-gel" kinase assay, which uses myelin basic protein as a substrate (lower part). Figure 6. The viability of HASMC after treatment with flavopiridol. Resting HASMC were treated with flavopiridol (75 nmol/l), TNF-α (50 ng/ml), or vehicle for the time periods indicated. Cell viability was assessed by trypan blue exclusion. The results are expressed as a percentage of viable cells in relation to the total cell count. Figure 7. Inhibition of neointimal formation in rat carotid artery by flavopiridol after balloon injury. The neointima/media ratios were measured in histological sections of rat carotid arteries treated with or without flavopiridol (5 mg/kg) for 5 days after injury. The arteries were examined 7 (n = 12) and 14 (n = 12) days after injury. The percentage of PCNA-positive nuclei (± SEM, expressed as a percentage of the number of nuclei counted) in the neointima of the arteries from each time point and treatment type is also given. * p < 0.05 compared to treatment with vehicle. Figure 8. Histological sections from rat carotid arteries. The sections are from the arteries 7 (A and B) and 14 (C and D) days after injury. The arteries shown in A and C are from rats treated with flavopiridol (5 mg/kg) by gavage; the arteries in B and D are from rats treated with vehicle alone. Original magnification x 100. Figure 9. Cdk2 expression after balloon injury in rat carotid arteries. The sections are four arteries 7 (A and B) and 14 (C and D) days after injury. The arteries shown in A and C are four rats treated with flavopiridol (5 mg/kg) by gavage; the arteries in B and D are from rats treated with vehicle alone. Cdk2-positive nuclei, located mainly in the neointima, are stained with vector blue according to the alkaline phosphatase method. Original magnification x 100.
Foreliggende oppfinnelse omfatter følgelig anvendelse av en forbindelse av formel Ia The present invention therefore encompasses the use of a compound of formula Ia
R5R5
I IN
OH O Formel Ia OH O Formula Ia
der Ri er hydrogen, Ci-C3-alkyl, naftyl, fenyl; fenyl mono- eller polysubstituert med halogen, Ci-C4-alkyl, Ci-C4-alkoksy, hydroksyl, karboksyl, COO-Ci-C6- alkyl, CONH2, CONH-C1-C6- alkyl, CON(Ci-C6-alkyl)2, nitro, trifluormetyl, amino, Ci-C4-alkylamino, di-Ci-C4-alkylamino, eller fenyl; pyridyl eller tienyl; where R 1 is hydrogen, C 1 -C 3 alkyl, naphthyl, phenyl; phenyl mono- or polysubstituted with halogen, Ci-C4-alkyl, Ci-C4-alkoxy, hydroxyl, carboxyl, COO-Ci-C6- alkyl, CONH2, CONH-C1-C6- alkyl, CON(Ci-C6-alkyl) 2, nitro, trifluoromethyl, amino, C 1 -C 4 -alkylamino, di-C 1 -C 4 -alkylamino, or phenyl; pyridyl or thienyl;
R2er hydrogen eller Ci-C3-alkyl; R 2 is hydrogen or C 1 -C 3 alkyl;
R5er Ci-C3-alkyl, C3-C5-sykloalkyl, eller C3-C5-sykloalkyl-Ci-C4-alkyl, R5 is C1-C3-alkyl, C3-C5-cycloalkyl, or C3-C5-cycloalkyl-C1-C4-alkyl,
eller et farmasøytisk akseptabelt syreaddisjonssalt derav, for fremstilling at et farmasøytisk preparat for inhibering av glattmuskel-proliferering, hvor dosen av forbindelsen i følge formel Ia er mindre enn 70 %, fortrinnsvis mindre enn 60 %, i or a pharmaceutically acceptable acid addition salt thereof, for the preparation of a pharmaceutical preparation for inhibiting smooth muscle proliferation, wherein the dose of the compound according to formula Ia is less than 70%, preferably less than 60%, in
særdeleshet mindre enn 50 % av doseringen som er nødvendig for å kontrollere tumorvekst. in particular less than 50% of the dosage required to control tumor growth.
Forbindelsene som anvendes ifølge oppfinnelsen har to asymmetriske sentre, ett der den heterosykliske ring inneholdende nitrogen er fusert til benzopyranenheten (C-4'), den The compounds used according to the invention have two asymmetric centers, one where the heterocyclic ring containing nitrogen is fused to the benzopyran unit (C-4'), the
andre ved karbonatom som bærer OH-gruppen på samme ring (C-3'), hvilket betyr at to par optiske isomerer er mulig. Definisjonen av forbindelsen ifølge oppfinnelsen omfatter alle mulige stereoisomerer og deres blandinger. Spesielt omfatter den racemiske former og de isolerte, optiske isomerer med spesiell aktivitet. De to racematene kan oppløses ved fysikalske metoder slik som for eksempel fraksjonskrystallisering. De enkelte optiske isomerer kan oppnås fra racematene ved konvensjonelle fremgangsmåter, slik som for eksempel saltdannelse med en optisk aktiv syre etterfulgt av krystallisering. Eksempel på alkylgrupper som er egnet for Ri til R5er rettkjedete eller forgrenede radikaler. others at the carbon atom bearing the OH group on the same ring (C-3'), which means that two pairs of optical isomers are possible. The definition of the compound according to the invention includes all possible stereoisomers and their mixtures. In particular, it includes racemic forms and the isolated optical isomers with special activity. The two racemates can be dissolved by physical methods such as, for example, fractional crystallization. The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization. Examples of alkyl groups suitable for R 1 to R 5 are straight-chain or branched radicals.
Egnede eksempler på salter av forbindelsene som anvendes ifølge oppfinnelsen med uorganiske eller organiske syrer er hydroklorid, hydrobromid, sulfat, fosfat, acetat, oksalat, tartrat, citrat, maleat eller fumarat. Suitable examples of salts of the compounds used according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulphate, phosphate, acetate, oxalate, tartrate, citrate, maleate or fumarate.
Særlig foretrukket er anvendelsen av forbindelser med formel Ia, der Ri er fenyl, tienyl, pyridyl, klorfenyl, diklorfenyl, metylfenyl, aminofenyl, bromfenyl, hydroksyfenyl eller naftyl; Particularly preferred is the use of compounds of formula Ia, where Ri is phenyl, thienyl, pyridyl, chlorophenyl, dichlorophenyl, methylphenyl, aminophenyl, bromophenyl, hydroxyphenyl or naphthyl;
R2er hydrogen og R 2 is hydrogen and
R5er metyl. R 5 is methyl.
En særlig viktig forbindelse er (-)-cis,-5,7-dihydroksy-2-(2-klorfenyl)-8-[4-(3-hydroksyl-metyl)-pipeirdinyl]-4H-benzopyran-4-one(Flavopiridol), spesielt i form av hydroklorid. A particularly important compound is (-)-cis,-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxyl-methyl)-pipeirdinyl]-4H-benzopyran-4-one ( Flavopiridol), especially in the form of hydrochloride.
Forbindelsene som anvendes ifølge oppfinnelsen kan fremstilles ifølge beskrivelsen i U.S. patent nr. 4 900 727 og U.S. patent nr. 5 284 856. Eksemplene i disse U.S. patenter er også viktige for foreliggende oppfinnelse. The compounds used according to the invention can be prepared according to the description in U.S. Pat. Patent No. 4,900,727 and U.S. Pat. Patent No. 5,284,856. The examples in these U.S. Pat. patents are also important for the present invention.
Forbindelsene som anvendes ifølge foreliggende oppfinnelse inhiberer glatt muskelcelleproliferering. De kan følgelig finne anvendelse som farmasøytiske preparater for inhibisjon av glatt muskelcelleproliferering, som inneholder minst en forbindelse med formel Ia, definert ovenfor eller minst ett av dens farmasøytisk akseptable syreaddisjonssalter. Typiske anvendelsesområder for forbindelsene som anvendes ifølge oppfinnelsen er sykdommer/forstyrrelser/skader forbundet med vaskulære lesjoner som er rike på glatte muskelceller. Et svært viktig eksempel er derfor lesjoner etter ballongskader. Et annet viktig anvendelsesområde er forebyggelse av restenose etter implantering av stenter. The compounds used according to the present invention inhibit smooth muscle cell proliferation. They may therefore find use as pharmaceutical preparations for inhibition of smooth muscle cell proliferation, containing at least one compound of formula Ia, defined above or at least one of its pharmaceutically acceptable acid addition salts. Typical areas of application for the compounds used according to the invention are diseases/disorders/injuries associated with vascular lesions that are rich in smooth muscle cells. A very important example is therefore lesions after balloon injuries. Another important area of application is the prevention of restenosis after implantation of stents.
For de farmasøytiske preparatene som oppnås ved oppfinnelsen anvendes en effektiv mengde av den nevnte aktive substans, enten pr. se eller eventuelt i kombinasjon med egnede farmasøytiske hjelpestoffer i form av tabletter, belagte tabletter, kapsler, stikkpiller, emulsjoner, suspensjoner eller løsninger, der innholdet av aktiv forbindelse er opptil 95 %, fortrinnsvis mellom 10 og 75 %. For the pharmaceutical preparations obtained by the invention, an effective amount of the aforementioned active substance is used, either per see or possibly in combination with suitable pharmaceutical excipients in the form of tablets, coated tablets, capsules, suppositories, emulsions, suspensions or solutions, where the content of active compound is up to 95%, preferably between 10 and 75%.
Fagpersonen vil vite hvilke hjelpestoffer som er egnede for den ønskede formuleringen av farmasøytisk preparat fordi dette ligger innenfor hans kunnskapsområde. Ved siden av hjelpestoffer for tabletter eller løsningsmidler, gelformer, baser for stikkpiller og andre eksipienser for den aktive substans, er det mulig og anvende for eksempel antioksidanter, dispergeringsmidler, emulgeringsmidler, skumhindrende midler, smaksstoffer, konserveringsmidler, oppløseliggjørende midler eller fargestoffer. The professional will know which excipients are suitable for the desired formulation of the pharmaceutical preparation because this is within his area of knowledge. In addition to auxiliaries for tablets or solvents, gel forms, bases for suppositories and other excipients for the active substance, it is possible to use, for example, antioxidants, dispersants, emulsifiers, anti-foaming agents, flavourings, preservatives, solubilizing agents or colourings.
Den aktive substans kan administreres oralt, parenteralt, intravenøst eller rektalt, oral administrering er fordelaktig. For en form for oral administrering kan den aktive substans blandes med andre forbindelser sammen med hjelpestoffene som er egnet i dette henseendet, slik som eksipienser, stabilisatorer og inerte fortynningsmidler, og vanlige fremgangsmåter kan anvendes for å overføre den til egnede administrasjonsformer, slik som tabletter, belagte tabletter, harde gelatinkapsler og vandige alkoholiske eller oljeformige suspensjoner eller løsninger. Eksempler på inerte eksipienser som kan anvendes er gummiarabicum, magnesiumoksid, laktose, glukose eller stivelse, i særdeleshet maisstivelse. I denne sammenheng kan formuleringen fremstilles som tørt granulat eller fuktig granulat. Eksempler på egnede oljeeksipienser eller løsningsmidler er vegetabilske eller animalske oljer, slik som solsikkeolje eller torskeleverolje. The active substance can be administered orally, parenterally, intravenously or rectally, oral administration being advantageous. For a form of oral administration, the active substance can be mixed with other compounds together with the excipients suitable in this regard, such as excipients, stabilizers and inert diluents, and conventional methods can be used to transfer it to suitable administration forms, such as tablets, coated tablets, hard gelatin capsules and aqueous alcoholic or oily suspensions or solutions. Examples of inert excipients that can be used are gum arabic, magnesium oxide, lactose, glucose or starch, in particular corn starch. In this context, the formulation can be prepared as dry granules or moist granules. Examples of suitable oil excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil.
Ved subkutan eller intravenøs administrering dannes en løsning, suspensjon eller emulsjon av den aktive substans, om nødvendig anvendes substanser som er konvensjonelle i denne hensikt, slik som oppløseliggjørende stoffer, emulgeringsmidler og andre hjelpestoffer. Eksempler på egnede fortynningsmidler er vann, fysiologisk natriumkloridløsning eller alkoholer, for eksempel etanol, propanol eller glyserol, og også sukkerløsninger slik som glykoseløsninger eller mannitolløsninger, eller en blanding av ulike løsningsmidler som er nevnt. With subcutaneous or intravenous administration, a solution, suspension or emulsion of the active substance is formed, if necessary, substances that are conventional for this purpose are used, such as solubilizing substances, emulsifiers and other auxiliary substances. Examples of suitable diluents are water, physiological sodium chloride solution or alcohols, for example ethanol, propanol or glycerol, and also sugar solutions such as glucose solutions or mannitol solutions, or a mixture of the various solvents mentioned.
Dosen av 4H-l-benzopyran-4-one-derivater som kan administreres daglig utvelges for å tilpasses den ønskede effekt. 4H-l-benzopyran-4-one-derivatene kan administreres i en dose som er mindre enn 70 %, fortrinnsvis mindre enn 60 %, i særdeleshet mindre enn 50 % av dosen, som anvendes for å kontrollere tumorvekst i det enkelte pattedyr. Et eksempel vil være - i en naken mus - xenograft modell - en dose på ca. 5 mg/kg kroppsvekt administrert oralt en gang daglig. Dette er halvparten av dosen som inhiberer tumorvekst i den samme dyremodell (Drees et al. Clin. Cancer Res. 1997; 3; 273-279). The dose of 4H-1-benzopyran-4-one derivatives that can be administered daily is selected to be adapted to the desired effect. The 4H-1-benzopyran-4-one derivatives can be administered in a dose that is less than 70%, preferably less than 60%, in particular less than 50% of the dose used to control tumor growth in the individual mammal. An example would be - in a nude mouse - xenograft model - a dose of approx. 5 mg/kg body weight administered orally once daily. This is half the dose that inhibits tumor growth in the same animal model (Drees et al. Clin. Cancer Res. 1997; 3; 273-279).
De farmakokinetiske egenskaper til 4H-l-benzopyran-4-one-derivatene kan gjøre det nødvendig å administrere forbindelsen flere ganger om dagen eller å velge langsomme frigivelsesformuleringer. The pharmacokinetic properties of the 4H-1-benzopyran-4-one derivatives may make it necessary to administer the compound several times a day or to choose slow release formulations.
Eksempler Examples
1. Flavopiridol inhiberer glatt muskelcelleproliferering og neointimal dannelse in vivo i en rotte carotismodell for vaskulær skade. 1. Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid model of vascular injury.
Den veletablerte rotte carotis skademodellen, der dannelse av neointimal lesjon etter kateterindusert skade er kritisk avhengig av SMC-proliferering (Clowes et al. Lab. Invest. 1983; 49:327-333, Clowes et al. Circ. Res. 1985; 56:139-145) ble anvendt for å undersøke om flavopiridol induserer vekststans i SMC in vivo, som det gjør in vitro. Flavopiridol ble administrert oralt ved en dose på 5 mg/kg en gang daglig, begynnende på skadedagen og forsatt 4 dager deretter, siden denne tidsperiode dekker den initielle induksjon av Cdk2 og den første bølge av SMC-proliferering i denne modell (Circ. Res. 1995; 77:445-465, Circ. Res. 1997; 80:418-426). Gjennomsnittlig intimalt og medialt areal ble kvantifisert 7 og 14 dager etter skaden, og neointimal lesjonsstørrelse ble uttrykt som forholdet mellom det neointimale og det mediale areal. Den behandlede og ubehandlede gruppen besto av 12 dyr. Forholdet på dag 7 var 1,00 ± 0,05 i arteriene hos vehikkelbehandlede rotter, og 0,65 ± 0,04 i flavopiridolbehandlede rottearterier, en reduksjon på 35 %. På dag 14 var det neointimale/mediale forholdet 1,08 ± 0,04 i vehikkelbehandlede rotter, og 0,66 ± 0,03 i flavopiridolbehandlede rotter, en reduksjon på 38,9 %. Disse effekter var statistisk signifikante ved begge tidspunkter (P < 0,05). The well-established rat carotid injury model, in which neointimal lesion formation following catheter-induced injury is critically dependent on SMC proliferation (Clowes et al. Lab. Invest. 1983; 49:327-333, Clowes et al. Circ. Res. 1985; 56: 139-145) was used to investigate whether flavopiridol induces growth arrest in SMC in vivo, as it does in vitro. Flavopiridol was administered orally at a dose of 5 mg/kg once daily, beginning on the day of injury and continuing for 4 days thereafter, since this time period covers the initial induction of Cdk2 and the first wave of SMC proliferation in this model (Circ. Res. 1995; 77:445-465, Circ. Res. 1997; 80:418-426). Mean intimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of neointimal to medial area. The treated and untreated group consisted of 12 animals. The ratio at day 7 was 1.00 ± 0.05 in the arteries of vehicle-treated rats, and 0.65 ± 0.04 in flavopiridol-treated rat arteries, a reduction of 35%. On day 14, the neointimal/medial ratio was 1.08 ± 0.04 in vehicle-treated rats, and 0.66 ± 0.03 in flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points (P < 0.05).
Metoder Methods
Materialer - Flavopiridol (L86-8275, (-)- cis, -5,7 -dihydroksy-2-(2-klorfenyl)-8-[4-(3-hydroksy-l-metyl)-piperidinyl]-4H-benzopyran-4-one) ble skaffet fra Hoechst Marion Roussel, Inc., og ble oppløst i dimetylsulfoksid som en stamløsning på 50 mmol/1 for cellekultureksperimenter eller i vann for in vivo eksperimenter. Basisk fibroblastvekstfaktor (bFGF) ble levert fra Collaborative Biochemical og trombin fra Sigma. MEK1-inhibitoren PD98059 ble skaffet fra New England Biolabs. Materials - Flavopiridol (L86-8275, (-)- cis, -5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-benzopyran -4-one) was obtained from Hoechst Marion Roussel, Inc., and was dissolved in dimethyl sulfoxide as a 50 mmol/l stock solution for cell culture experiments or in water for in vivo experiments. Basic fibroblast growth factor (bFGF) was supplied from Collaborative Biochemical and thrombin from Sigma. The MEK1 inhibitor PD98059 was obtained from New England Biolabs.
Cellekultur - Glatte muskelceller fra human aorta (HASMC) ble skaffet fra Clonetics og ble dyrket som tidligere beskrevet<18>. Celler ble anvendt ved passasjene 5-9. Før eksperimentene ble utført ble veksten stoppet ved 80 % konfluens i 48 timer med medium inneholdende 0,2 % føtalt bovint serum. Cell culture - Human aortic smooth muscle cells (HASMC) were obtained from Clonetics and were cultured as previously described<18>. Cells were used at passages 5-9. Before experiments were performed, growth was arrested at 80% confluence for 48 h with medium containing 0.2% fetal bovine serum.
Celleprolifererings ELISA - Celleproliferasjon ble målt ved ELISA (Amersham Life Science). HASMC ble dyrket i gelatinbelagte 96-brønns plater og gjort hvilende. Celler ble behandlet med 10 ng/ml bFGF, 2 U/ml trombin, eller vehikkel i 24 timer. Flavopiridol (75 nmol/1) ble administrert 1 time før vekstfaktorbehandling. 5-brom-2'-deoksyuridin (BrdU) ble tilsatt til en sluttkonsentrasjon på 10 pmol/1 under de 2 siste timene av eksperimentet. BrdU-inkorporering ble målt som beskrevet<19>. Resultatene uttrykkes som gjennomsnitt ± standardfeil for 12 prøver pr. betingelse for to uavhengige eksperimenter. Cell proliferation ELISA - Cell proliferation was measured by ELISA (Amersham Life Science). HASMC were cultured in gelatin-coated 96-well plates and made quiescent. Cells were treated with 10 ng/ml bFGF, 2 U/ml thrombin, or vehicle for 24 h. Flavopiridol (75 nmol/l) was administered 1 hour before growth factor treatment. 5-bromo-2'-deoxyuridine (BrdU) was added to a final concentration of 10 pmol/l during the last 2 hours of the experiment. BrdU incorporation was measured as described<19>. The results are expressed as mean ± standard error for 12 samples per condition for two independent experiments.
Celleantall - Vekststansede HASMC, dyrket til 50 % konfluens i 6-brønns plater ble behandlet med eller uten flavopiridol (75 nmol/1) eller bFGF (10 ng/ml). Ved intervaller etter behandling ble celler trypsinert og celleantall bestemt ved anvendelse av et hemocytometer. Cell Count - Arrested HASMCs, grown to 50% confluence in 6-well plates were treated with or without flavopiridol (75 nmol/1) or bFGF (10 ng/ml). At intervals after treatment, cells were trypsinized and cell counts determined using a hemocytometer.
Western blot analyser - Hvilende HASMC ble behandlet i nærvær eller fravær av vekstfaktorer og/eller flavopiridol som antydet. Western blot analyser ble utført som tidligere beskrevet<18>. De primære antistoffer var: Et polyklonalt antihumant syklin Dl-antistoff (M-20, Santa Cruz), et monoklonalt antihumant prolifererende cellekjerne antigen (PCNA)-antistoff (PC 10, Sigma), et fosforyleringsspesifikt p44/42 (Erkl/Erk2) MAP-kinase monoklonalt antistoff (New England Biolabs), og et monoklonalt anti-Rb-antistoff (G3-245, Pharmigen), som gjenkjenner de fosforylerte (pRb) og de høyt fosforylerte (ppRb) Rb-typene. For immunblottingsstudier ble eksperimentet gjentatt minst tre ganger. Western blot analyzes - Resting HASMC were treated in the presence or absence of growth factors and/or flavopiridol as indicated. Western blot analyzes were performed as previously described<18>. The primary antibodies were: A polyclonal antihuman cyclin D1 antibody (M-20, Santa Cruz), a monoclonal antihuman proliferating cell nuclear antigen (PCNA) antibody (PC 10, Sigma), a phosphorylation-specific p44/42 (Erk1/Erk2) MAP -kinase monoclonal antibody (New England Biolabs), and an anti-Rb monoclonal antibody (G3-245, Pharmigen), which recognizes the phosphorylated (pRb) and the highly phosphorylated (ppRb) Rb types. For immunoblotting studies, the experiment was repeated at least three times.
Cdk- aktivitet - Hvilende HASMC ble behandlet med agonister og inhibitorer i 24 timer og totalt cellelysat ble tilberedt som beskrevet for Western blotting. Kinaseanalysen ble utført med et histon og et kinase-analysesett (Upstate Biotechnology) ifølge produsentens bruksanvisning. I korthet ble 10 pl peptidinhibitorer for protein kinase C (2 pmol/1) og protein kinase A (2 pmol/1), 100 ug cellelysat, 10 jol analysebuffer og 10 pl av en blanding inneholdende 75 pmol/1 magnesiumklorid, 500 pmol/1 ATP og 1 pCi/ml [y -<32>P]ATP blandet i et mikrosentrifugerør. Etter inkubering ved 30 °C i 10 minutter ble alikvoter pipettert på fosforcellulosepapir. Papirene ble vasket i 0,75 % fosforsyre, etterfulgt av målinger av cpm i en scintillasjonsteller (Beckman). Resultatene uttrykkes som gjennomsnitt ± standardfeil for 3 prøver og er representative for tre uavhengige eksperimenter. Cdk activity - Resting HASMC were treated with agonists and inhibitors for 24 h and total cell lysate was prepared as described for Western blotting. The kinase assay was performed with a histone and kinase assay kit (Upstate Biotechnology) according to the manufacturer's instructions. Briefly, 10 µl of peptide inhibitors for protein kinase C (2 pmol/1) and protein kinase A (2 pmol/1), 100 µg of cell lysate, 10 µl of assay buffer and 10 µl of a mixture containing 75 pmol/1 magnesium chloride, 500 pmol/ 1 ATP and 1 pCi/ml [γ -<32>P]ATP mixed in a microcentrifuge tube. After incubation at 30°C for 10 minutes, aliquots were pipetted onto phosphor cellulose paper. The papers were washed in 0.75% phosphoric acid, followed by measurements of cpm in a scintillation counter (Beckman). Results are expressed as mean ± standard error of 3 samples and are representative of three independent experiments.
" In- gel"- kinaseanalyse - Hvilende HASMC ble behandlet med vekstfaktorer i 30 minutter og totalt cellelysat ble tilberedt som beskrevet for Western blotting. I noen eksperimenter ble HASMC forbehandlet i 60 minutter med 30 nmol/1 PD98059, flavopiridol eller vehikkel. Like mengder av proteiner (50 ng/felt) ble oppløst på en polyakrylamidgel som ble ko-polymerisert med 350 ng/ml basisk myelinprotein. Gelen ble behandlet med[y-32P]ATP og autoradiografi ble utført som beskrevet<19>. "In-Gel" Kinase Assay - Resting HASMC were treated with growth factors for 30 minutes and total cell lysate was prepared as described for Western blotting. In some experiments, HASMC were pretreated for 60 min with 30 nmol/l PD98059, flavopiridol, or vehicle. Equal amounts of proteins (50 ng/field) were resolved on a polyacrylamide gel that was copolymerized with 350 ng/ml basic myelin protein. The gel was treated with [γ-32P]ATP and autoradiography was performed as described<19>.
Trypanblå- eksklusjon - HASMC ble dyrket i 5-cm skåler ved lav konfluens og veksten arrestert som beskrevet. Celler ble behandlet med flavopiridol (75 nmol/1) eller tumornekrosefaktor-a (TNF-a; 50 ng/ml) for de antydede tidspunkter. Etter fjerning av mediet ble 0,4 % trypanblått i fosfatbufret saltvann tilsatt skålene. Etter 5 minutter ble cellene i skålen telt under mikroskop. Blå celler ble telt som ikke levedyktige celler. Trypan blue exclusion - HASMC were grown in 5-cm dishes at low confluence and growth arrested as described. Cells were treated with flavopiridol (75 nmol/l) or tumor necrosis factor-α (TNF-α; 50 ng/ml) for the indicated time points. After removal of the medium, 0.4% trypan blue in phosphate-buffered saline was added to the dishes. After 5 minutes, the cells in the dish were counted under a microscope. Blue cells were counted as non-viable cells.
Rotte carotis skademodell - Skade på rotte carotisarterien ble utført i hovedsak som beskrevet . Voksne Sprague-Dawley hannrotter (400-500 g, Zivic-Miller) ble anestesert med en intraperitoneal injeksjon av ketamin (2 mg/kg) og xylazin (4 mg/kg). Venstre carotis interna ble deretter kanylert med et 2F embolectomikateter. Ballongen ble blåst opp med saltvann og trukket gjennom arterien tre ganger for å frembringe en utvidende og blottleggende skade. Den høyre carotisarterien forble uskadet og fungerte som en kontroll på skade for hvert dyr. Umiddelbart etter rekonstitusjon etter anestesi og i 4 påfølgende dager ble rottene administrert med flavopiridol (5 mg/kg i vann) eller vann ved sondeforing som blindtest. Alle rottene overlevde kirurgien og det var ingen tydelige tegn på toksisitet forbundet med medikamentadministreringen i de anvendte dosene. Ved særskilte tidspunkter etter carotisskaden ble rottene anestesert som ovenfor og perfusjonsfiksert systemisk med 4 % paraformaldehyd i fosfatbufret saltvann. Høyre og venstre carotisarterier ble fjernet og utspilt ved injeksjon av 4 % paraformaldehyd gjennom lumen, hvorpå de ble dehydrert og lagret i 70 % etanol ved 4 °C. Immunhistokjemi ble utført som beskrevet tidligere<18>, ved anvendelse av det monoklonale PCNA-antistoff og et polyklonalt antihumant Cdk2-antistoff (M2-G, Santa Cruz). Rat carotid injury model - Injury to the rat carotid artery was performed essentially as described. Adult male Sprague-Dawley rats (400-500 g, Zivic-Miller) were anesthetized with an intraperitoneal injection of ketamine (2 mg/kg) and xylazine (4 mg/kg). The left internal carotid was then cannulated with a 2F embolectomy catheter. The balloon was inflated with saline and pulled through the artery three times to produce an expanding and exposing lesion. The right carotid artery remained uninjured and served as an injury control for each animal. Immediately after reconstitution from anesthesia and for 4 consecutive days, the rats were administered flavopiridol (5 mg/kg in water) or water by gavage as a blind test. All the rats survived the surgery and there were no obvious signs of toxicity associated with the drug administration at the doses used. At separate times after the carotid injury, the rats were anesthetized as above and perfusion-fixed systemically with 4% paraformaldehyde in phosphate-buffered saline. Right and left carotid arteries were removed and distended by injection of 4% paraformaldehyde through the lumen, after which they were dehydrated and stored in 70% ethanol at 4 °C. Immunohistochemistry was performed as described previously<18>, using the monoclonal PCNA antibody and a polyclonal antihuman Cdk2 antibody (M2-G, Santa Cruz).
Billedanalyse - De ytterste distale og proksimale områder av hver arterie (ca. 500 pm) ble fjernet. Ti mellomliggende tverrsnitt (8 pm hver) tatt med 500 pm mellomrom ble analysert for hver arterie. Snitt ble fiksert og farget med hematoxilin og eosin som tidligere beskrevet<18>. Ved anvendelse av et Nikon Diaphot 300 mikroskop og et 4x objektiv, ble hvert tverrsnitt tatt vare på som et digitalt bilde ved hjelp av et Hamamatsu C5985 videokamera og TCPro 2,41 (Coreco, Inc.). Mediale og neointimale områder ble bestemt ved anvendelse av NIH Image programvare. Mediale og neointimale grenser ble bestemt av en snittavleser (A.M.) og bekreftet på en blind måte av en annen avleser (C.P.). Lesjonsstørrelsen ble uttrykt som neointima/media-forholdet. Resultater for hver gruppe ble uttrykt som gjennomsnitt ± standardfeil. 92 % eller mer av bildene kunne tydes i hver gruppe; de resterende ble ikke analysert pga. fikseringsartifakter. Image Analysis - The outermost distal and proximal regions of each artery (approximately 500 pm) were removed. Ten intermediate cross-sections (8 µm each) taken at 500 µm intervals were analyzed for each artery. Sections were fixed and stained with hematoxylin and eosin as previously described<18>. Using a Nikon Diaphot 300 microscope and a 4x objective, each cross-section was captured as a digital image using a Hamamatsu C5985 video camera and TCPro 2.41 (Coreco, Inc.). Medial and neointimal areas were determined using NIH Image software. Medial and neointimal borders were determined by one section reader (A.M.) and confirmed in a blinded manner by another reader (C.P.). Lesion size was expressed as the neointima/media ratio. Results for each group were expressed as mean ± standard error. 92% or more of the images could be deciphered in each group; the rest were not analyzed because fixation artifacts.
Statistiske analyser - Dersom det var hensiktsmessig ble data fra kvantitative studier uttrykt som gjennomsnitt ± standardfeil. For multiple behandlingsgrupper ble en faktoriell ANOVA etterfulgt av Fisher's minste signifikans forskjellstest anvendt. Statistisk signifikans ble akseptert ved p < 0,05. Statistical analyzes - If appropriate, data from quantitative studies were expressed as mean ± standard error. For multiple treatment groups, a factorial ANOVA followed by Fisher's least significant difference test was used. Statistical significance was accepted at p < 0.05.
Resultater Results
Flavopiridol inhiberer HASMC- proliferering - På bakgrunn av flavopiridols evne til å inhibere proliferasjon av ulike tumorcellelinjer, ble hypotesen om at dens anvendelse ville blokkere vekst av primærkultur av humane SMC undersøkt. Vekststansede HASMC ble behandlet med SMC-mitogenet bFGF (10 ng/ml) i 24 timer i nærvær av økende konsentrasjoner av flavopiridol og proliferasjonen ble målt ved en ELISA-basert analyse. Sammenliknet med ubehandlede celler økte proliferasjonen av bFGF-behandlede celler 5,4 ganger (figur IA). Forbehandling i 1 time med så lite som 50 nmol/1 flavopiridol reduserte HASMC-proliferasjonen betydelig (til 3,9-ganger,/> < 0,05), en effekt som var omtrent maksimal ved konsentrasjoner på 75 nmol/1. Tilsvarende resultater ble oppnådd ved anvendelse av tymidinopptak som et uavhengig mål på DNA-syntese (ikke vist). Flavopiridol inhibits HASMC proliferation - On the basis of flavopiridol's ability to inhibit proliferation of various tumor cell lines, the hypothesis that its use would block the growth of primary culture of human SMCs was investigated. Growth-arrested HASMCs were treated with the SMC mitogen bFGF (10 ng/ml) for 24 h in the presence of increasing concentrations of flavopiridol and proliferation was measured by an ELISA-based assay. Compared to untreated cells, the proliferation of bFGF-treated cells increased 5.4-fold (Figure 1A). Pretreatment for 1 hour with as little as 50 nmol/1 flavopiridol significantly reduced HASMC proliferation (to 3.9-fold, /> < 0.05), an effect that was approximately maximal at concentrations of 75 nmol/1. Similar results were obtained using thymidine uptake as an independent measure of DNA synthesis (not shown).
For å undersøke alminneligheten i flavopiridols effekt på SMC-proliferasjon, ble dens effekt på mitogenese fremkalt av trombin (2 U7ml), som virker gjennom en G-proteinkoplende reseptor, tilsvarende bFGF, som stimulerer et medlem av reseptortyrosinkinasefamilien undersøkt. Flavopiridol (75 nmol/1) inhiberte signifikant og kraftig, både bFGF- og trombinindusert HASMC- proliferering (henholdsvis 5,4-ganger vs. 1,8-ganger og 2,4-ganger vs. 0,7-ganger, p < 0,05, figur IB). Det ble utført celletellinger for å bekrefte at effekten av flavopiridol på cellesyklusprogresjonen i HASMC virkelig gjenspeilet endringer i proliferasjon. bFGF (10 ng/ml) induserte en 3-gangers økning i celleantall etter 3 dagers behandling (figur 2). Tilsvarende resultater ble vist i de ELISA-baserte analyser, flavopiridol (75 nmol/1) blokkerte effektivt bFGF-indusert proliferering. To examine the generality of flavopiridol's effect on SMC proliferation, its effect on mitogenesis elicited by thrombin (2 U7ml), which acts through a G protein-coupled receptor, similar to bFGF, which stimulates a member of the receptor tyrosine kinase family, was examined. Flavopiridol (75 nmol/1) significantly and potently inhibited both bFGF- and thrombin-induced HASMC proliferation (5.4-fold vs. 1.8-fold and 2.4-fold vs. 0.7-fold, respectively, p < 0.05, Figure 1B). Cell counts were performed to confirm that the effect of flavopiridol on cell cycle progression in HASMCs indeed reflected changes in proliferation. bFGF (10 ng/ml) induced a 3-fold increase in cell number after 3 days of treatment (Figure 2). Corresponding results were shown in the ELISA-based assays, flavopiridol (75 nmol/1) effectively blocked bFGF-induced proliferation.
Flavopiridol inhiberer Cdk- aktivitet og cellesyklusrelatert genekspresjon i HASMC - For å vurdere den spesifikke effekt til flavopiridol på cellesyklusmaskineriet, ble histon Hl-kinaseaktiviteten i cellelysater fra vekstfaktorstimulerte HASMC målt. Fosforylering av histon Hl gjenspeiler aktivitetene til Cdc2og Cdk2<20>. Behandling av HASMC med bFGF og trombin resulterte i henholdsvis 4,4-gangers og 3,6-gangers økning i histon Hl-kinaseaktivitet (figur 3). Disse økninger i syklinavhengig kinaseaktivitet ble totalt blokkert ved forbehandling med flavopiridol (75 nmol/1). Flavopiridol inhibits Cdk activity and cell cycle-related gene expression in HASMC - To assess the specific effect of flavopiridol on the cell cycle machinery, the histone H1 kinase activity in cell lysates from growth factor-stimulated HASMC was measured. Phosphorylation of histone Hl reflects the activities of Cdc2 and Cdk2<20>. Treatment of HASMC with bFGF and thrombin resulted in 4.4-fold and 3.6-fold increases in histone H1 kinase activity, respectively (Figure 3). These increases in cyclin-dependent kinase activity were completely blocked by pretreatment with flavopiridol (75 nmol/1).
Ved Western blot analyse ble det også undersøkt om flavopiridol påvirket Western blot analysis also investigated whether flavopiridol was affected
vekstfaktorindusert regulering av cellesyklusrelaterte proteiner i HASMC. Syklin Di er et Gi-fase syklin som oppreguleres ved vekstfaktorstimulering og som raskt degraderes ved fjerning fra cellesyklus 21. Syklin Di-proteinnivåer ble oppregulert, henholdsvis 6,3-ganger og 3,2-ganger som respons på bFGF- og trombinbehandling i 24 timer (figur 4), en effekt som fullstendig kunne blokkeres ved forbehandling med flavopiridol. Tilsvarende ble økning i ekspresjon av PCNA, som hovedsakelig syntetiseres under S-fasen i forbindelse med DNA-replikasjon<22>, også blokkert ved flavopiridol forbehandling. Som et endelig mål på cellesyklusrelaterte proteiner ble Rb-fosforylering som respons på vekstfaktorekspresjon ved anvendelse av et antistoff som gjenkjenner fosforylert Rb undersøkt. Rb er en cellesyklusregulator som bindes til, og inaktiverer, transkripsjonsfaktoren E2F når Rb er i ufosforylert tilstand<23>og induserer SMC-vekststans in vivo n. Fosforylering inaktiverer Rb og tillater at progresjonen gjennom S-fasen fortsetter. Analyse av Rb-fosforylering er særlig relevant fordi Rb er et mål for Cdk2og Cdktin vivo. Både trombin- og bFGF-indusert hyperfosforylering av Rb var en effekt som ble inhibert av flavopiridol. Totalt antyder disse resultater at flavopiridol growth factor-induced regulation of cell cycle-related proteins in HASMC. Cyclin Di is a Gi-phase cyclin that is upregulated upon growth factor stimulation and rapidly degraded upon removal from the cell cycle 21. Cyclin Di protein levels were upregulated, 6.3-fold and 3.2-fold, respectively, in response to bFGF and thrombin treatment for 24 hours (Figure 4), an effect that could be completely blocked by pretreatment with flavopiridol. Similarly, increase in expression of PCNA, which is mainly synthesized during S-phase in connection with DNA replication<22>, was also blocked by flavopiridol pretreatment. As a final measure of cell cycle-related proteins, Rb phosphorylation in response to growth factor expression using an antibody that recognizes phosphorylated Rb was examined. Rb is a cell cycle regulator that binds to, and inactivates, the transcription factor E2F when Rb is in an unphosphorylated state<23>and induces SMC growth arrest in vivo n. Phosphorylation inactivates Rb and allows progression through S phase to continue. Analysis of Rb phosphorylation is particularly relevant because Rb is a target for Cdk2 and Cdktin in vivo. Both thrombin- and bFGF-induced hyperphosphorylation of Rb was an effect that was inhibited by flavopiridol. Overall, these results suggest that flavopiridol
påvirker ekspresjon og aktivitet av Gi og S-faserelaterte cellesykluskontrollelementer i HASMC forbundet med dens vekstinhibitoriske effekter. affects expression and activity of Gi and S-phase-related cell cycle control elements in HASMC associated with its growth inhibitory effects.
Flavopiridol har ingen effekt på MAP- kinasefosforylering eller - aktivitet - For å sikre at flavopiridol virker spesielt på cellesyklusnivået, heller enn uspesifikt på oppstrøms kinase-reaksjonsveier, ble fosforylering og aktivitet av Erkl (p44 MAP-kinase) og Erk2 (p42 MAP-kinase), to medlemmer av MAP-kinasefamilien, målt. Disse kinaser ble valgt fordi de befinner seg umiddelbart oppstrøms for transkripsjonshendelsene som oppstår som respons på vekststimuli og nedstrøms for et antall kritiske mitogene signalreaksjonsveier<24>. En intakt respons av MAP kinaser antyder at de oppstrøms mitogene reaksjonsveier også er intakte. Fosforyleringsstatusen til Erkl og Erk2 ble målt med et monoklonalt antistoff som spesifikt gjenkjenner fosforyleringen - og - følgelig aktiverte former. Som en kontroll på disse eksperimenter ble PD98059, en potent og selektiv inhibitor for MAP kinaseaktiviteten, anvendt 25. Økte mengder av fosforylert Erkl og Erk2 ble påvist etter behandling av HASMC med trombin og bFGF Flavopiridol has no effect on MAP kinase phosphorylation or activity - To ensure that flavopiridol acts specifically at the cell cycle level, rather than nonspecifically on upstream kinase pathways, phosphorylation and activity of Erk1 (p44 MAP kinase) and Erk2 (p42 MAP kinase ), two members of the MAP kinase family, measured. These kinases were chosen because they are located immediately upstream of the transcriptional events that occur in response to growth stimuli and downstream of a number of critical mitogenic signaling pathways<24>. An intact response of MAP kinases suggests that the upstream mitogenic pathways are also intact. The phosphorylation status of Erk1 and Erk2 was measured with a monoclonal antibody that specifically recognizes the phosphorylation - and - consequently activated forms. As a control for these experiments, PD98059, a potent and selective inhibitor of MAP kinase activity, was used 25. Increased amounts of phosphorylated Erk1 and Erk2 were detected after treatment of HASMC with thrombin and bFGF
i 30 minutter, sammenliknet med ubehandlede celler (figur 5, øvre del). Fosforylering av Erkl og Erk 2 ved både trombin og bFGF ble blokkert ved forbehandling med PD98059, men ikke med flavopiridol. For å bekrefte disse funn ble Erkl - og Erk2-aktivitet målt ved en "in-gel" kinaseanalyse (figur 5, nedre del). Igjen ble det funnet at Erkl - og Erk2-aktivitetene økte som respons på trombin og bFGF, en effekt som kunne inhiberes av PD98059, men ikke av flavopiridol. Disse eksperimenter sammen med de som er representert i figur 3 og 4, frembringer bevis for at effektene av flavopiridol på HASMC-proliferering er forårsaket av en spesifikk arrest av cellesyklusmaskineriet ved blokkering av Cdk-aktiviteten uten å påvirke oppstrøms signaliseringshendelser. for 30 min, compared to untreated cells (Figure 5, upper part). Phosphorylation of Erk1 and Erk2 by both thrombin and bFGF was blocked by pretreatment with PD98059, but not with flavopiridol. To confirm these findings, Erk1 and Erk2 activity was measured by an "in-gel" kinase assay (Figure 5, lower part). Again, Erk1 and Erk2 activities were found to increase in response to thrombin and bFGF, an effect that could be inhibited by PD98059 but not by flavopiridol. These experiments along with those represented in Figures 3 and 4 provide evidence that the effects of flavopiridol on HASMC proliferation are caused by a specific arrest of the cell cycle machinery by blocking Cdk activity without affecting upstream signaling events.
Flavopiridol øker ikke HASMC- levedyktigheten - Tidligere rapporter vedrørende flavopiridolaktivitet i andre celletyper har vist at, avhengig av celletypen, kan flavopiridol enten indusere vekststans uten å påvirke levedyktigheten, eller den kan forårsake apoptose<16>'<26>'<29>. Det ble derfor vurdert om flavopiridol reduserte levedyktigheten til HASMC ved å måle trypanblått-eksklusjon ved ulike tidspunkter etter behandling. Hvilende HASMC ble behandlet med flavopiridol (75 nmol/1), vehikkel eller TNF-a (50 ng/ml), et cytokin kjent for å indusere apoptose i denne celletype<30>. Mens TNF-a reduserer levedyktigheten til HASMC kraftig, hvilket resulterer i død av hovedsakelig alle celler behandlet i 24 timer, hadde flavopiridol ingen slik effekt (figur 6). Det ble observert at med høyere konsentrasjoner og lengre inkuberinger kunne noe reduksjon i levedyktighet i nærvær av flavopiridol forekomme Flavopiridol does not increase HASMC viability - Previous reports regarding flavopiridol activity in other cell types have shown that, depending on the cell type, flavopiridol can either induce growth arrest without affecting viability, or it can cause apoptosis<16>'<26>'<29>. It was therefore assessed whether flavopiridol reduced the viability of HASMC by measuring trypan blue exclusion at different time points after treatment. Resting HASMC were treated with flavopiridol (75 nmol/1), vehicle or TNF-α (50 ng/ml), a cytokine known to induce apoptosis in this cell type<30>. While TNF-α strongly reduces the viability of HASMC, resulting in the death of essentially all cells treated for 24 h, flavopiridol had no such effect (Figure 6). It was observed that with higher concentrations and longer incubations some reduction in viability in the presence of flavopiridol could occur
(ikke vist). Imidlertid, under de undersøkte betingelser induserer flavopiridol hovedsakelig vekststans, uten å påvirke SMC-levedyktigheten. (not shown). However, under the conditions investigated, flavopiridol mainly induces growth arrest, without affecting SMC viability.
Flavopiridol inhiberer glatt muskelcelleproliferering og neointimal dannelse in vivo i en rotte carotis skademodell på vaskulær skade - Den veletablerte rotte carotis skademodell ble anvendt der dannelse av neointimale lesjoner etter kateterindusert skade er kritisk avhengig av SMC-proliferering<2>'<3>, for å undersøke om flavopiridol induserer vekststans av SMC in vivo, slik det gjør in vitro. Flavopiridol ble administrert oralt ved en dose på 5 mg/kg en gang daglig, begynnende på skadedagen og fortsatte de 4 neste dagene, siden denne tidsperiode dekker den initielle injeksjon av Cdk2og den første bølge av SMC-proliferering i denne modell<31>'<32>. Gje<n>nomsnittlige intimale og medialarealer ble kvantifisert 7 og 14 dager etter skade, og neointimal lesjonsstørrelse ble uttrykt som forholdet mellom det neointimale areal og det mediale areal. Det var 12 dyr i både den behandlede og ubehandlede gruppe. Det neointimale/mediale forholdet ved 7 dager var 1,00 ± 0,05 i arteriene hos vehikkelbehandlede rotter, og 0,65 ± 0,04 i flavopiridolbehandlede rottearterier, en reduksjon på 35,0 % (figur 7). På dag 14 var det neointimale/mediale forhold 1,08 ± 0,04 i vehikkelbehandlede rotter, og 0,66 ± 0,03 i flavopiridolbehandlede rotter, en reduksjon på 38,9 %. Disse effekter var statistisk signifikante ved begge tidspunkter ( p < 0,05). Representative arteriesnitt er vist i figur 8. Flavopiridol inhibits smooth muscle cell proliferation and neointimal formation in vivo in a rat carotid injury model of vascular injury - The well-established rat carotid injury model was used in which formation of neointimal lesions after catheter-induced injury is critically dependent on SMC proliferation<2>'<3>, to investigate whether flavopiridol induces growth arrest of SMC in vivo, as it does in vitro. Flavopiridol was administered orally at a dose of 5 mg/kg once daily, beginning on the day of injury and continuing for the next 4 days, since this time period covers the initial injection of Cdk2 and the first wave of SMC proliferation in this model<31>'< 32>. Average intimal and medial areas were quantified 7 and 14 days after injury, and neointimal lesion size was expressed as the ratio of the neointimal area to the medial area. There were 12 animals in both the treated and untreated groups. The neointimal/medial ratio at 7 days was 1.00 ± 0.05 in the arteries of vehicle-treated rats, and 0.65 ± 0.04 in flavopiridol-treated rat arteries, a reduction of 35.0% (Figure 7). On day 14, the neointimal/medial ratio was 1.08 ± 0.04 in vehicle-treated rats, and 0.66 ± 0.03 in flavopiridol-treated rats, a reduction of 38.9%. These effects were statistically significant at both time points ( p < 0.05). Representative artery sections are shown in Figure 8.
For direkte å demonstrere at flavopiridol inhiberer glatt muskelcelleproliferering, ble snitt farget for PCNA-ekspresjon i representative områder fira hver arterie og prosentandelen av PCNA-positive kjerner i neointima ble bestemt. På dag 7 var 31,1 7,2 % av kjernene i skadede områder fra ubehandlede rotter PCNA-positive, mens bare 11,8 ± 1,5 % av skadete arterier i flavopiridolbehandlede rotter var PCNA-positive (figur l;p < 0,05). På dag 14 var PCNA-positive kjerner til stede i 10,4 ± 2,0 % av neointimale celler fra behandlede, men bare i 4,2 ± 0,5 % av neointimale celler fra ubehandlede, uskadde rottearterier ( p < 0,05). (PCNA-positive kjerner ble sjelden sett i uskadde arterier, uavhengig av behandling.) Tilsvarende var Cdk2-positive celler langt mindre vanlige i neointima av flavopiridolbehandlede rotter (figur 9, A og Q, sammenliknet med arterier fra ubehandlede rotter ( B og D), ved både 7 og 14 dager etter skade. To directly demonstrate that flavopiridol inhibits smooth muscle cell proliferation, sections were stained for PCNA expression in representative areas of each artery and the percentage of PCNA-positive nuclei in the neointima was determined. On day 7, 31.1 7.2% of cores in injured areas from untreated rats were PCNA-positive, whereas only 11.8 ± 1.5% of injured arteries in flavopiridol-treated rats were PCNA-positive (Figure 1; p < 0 .05). At day 14, PCNA-positive nuclei were present in 10.4 ± 2.0% of neointimal cells from treated but only in 4.2 ± 0.5% of neointimal cells from untreated, uninjured rat arteries ( p < 0.05 ). (PCNA-positive nuclei were rarely seen in uninjured arteries, regardless of treatment.) Similarly, Cdk2-positive cells were far less common in the neointima of flavopiridol-treated rats (Figure 9, A and Q, compared with arteries from untreated rats (B and D) , at both 7 and 14 days after injury.
Diskusjon Discussion
I foreliggende studie er det undersøkt om den nye Cdk-inhibitoren, flavopiridol, den mest potente og spesifikke inhibitor av Cdk som er kjent, er en egnet kandidat for å inhibere SMC-proliferering in vivo, spesielt innenfor området vaskulær skade. Tidligere forsøk på å styre cellesyklusmaskineriet terapeutisk ved behandling av vaskulære sykdommer har frembrakt et rasjonale for de foreliggende studier<9>"<12>; imidlertid er fremgangsmåter som frem til nå er anvendt bygget på genoverføringsteknologi for å inhibere cellesyklusprogresjon. Store kliniske vanskeligheter hindrer for tiden anvendelse av disse teknikker<33>. Under disse studier ble det rapportert at CVT-313, en nylig identifisert forbindelse som også har Cdk-inhibitoriske egenskaper, men ved mikromolare konsentrasjoner, også kan inhibere neointimal dannelse; imidlertid var det nødvendig at CVT-313 ble tilført carotisarterien på skadetidspunktet for å frembringe denne effekt<34>.1 motsetning ble det vist at oralt administrert flavopiridol, kraftig kan inhibere intimal dannelse til et nivå sammenliknbart med andre klinisk relevante midler<n>'<35>'<36>. Den orale aktivitet til flavopiridol gjør den unik blant midler som har blitt vist å være aktive i dyremodeller på vaskulær skade. Dens selektivitet, styrke og enkle administrering gjør flavopiridol til en utmerket kandidat for å undersøke de terapeutiske fordeler ved cellesyklusinhibisjon in vivo ved humane, vaskulære lesjoner. In the present study, it has been investigated whether the new Cdk inhibitor, flavopiridol, the most potent and specific inhibitor of Cdk known, is a suitable candidate for inhibiting SMC proliferation in vivo, especially in the area of vascular damage. Previous attempts to control the cell cycle machinery therapeutically in the treatment of vascular diseases have produced a rationale for the present studies<9>"<12>; however, methods used until now are based on gene transfer technology to inhibit cell cycle progression. Major clinical difficulties prevent for time application of these techniques<33>. During these studies, it was reported that CVT-313, a newly identified compound that also has Cdk-inhibitory properties, but at micromolar concentrations, can also inhibit neointimal formation; however, it was necessary that CVT- 313 was infused into the carotid artery at the time of injury to produce this effect<34>. In contrast, it was shown that orally administered flavopiridol can strongly inhibit intimal formation to a level comparable to other clinically relevant agents<n>'<35>'<36> The oral activity of flavopiridol makes it unique among agents that have been shown to be active in animal models of vascular r damage. Its selectivity, potency and ease of administration make flavopiridol an excellent candidate for investigating the therapeutic benefits of cell cycle inhibition in vivo in human vascular lesions.
Det ble valgt å administrere flavopiridol oralt i en konsentrasjon på 5 mg/kg, halvparten av den dose som inhiberer tumorvekst i en naken mus xenograft modell 21. Det er bemerkelsesverdig at flavopiridolkonsentrasjoner på 75 nmol/1 resulterer i tilnærmet fullstendig inhibisjon av SMC-proliferasjon i foreliggende studier, mens midlere serumkonsentrasjoner på 425 nmol/1 ble oppnådd ved doser under den toksiske grense i fase 1 humane studier på refraktært karsinom<17>. Foreliggende resultater antyder at langt lavere doser av cellesyklusinhibitorer enn de som anvendes ved neoplasier, kan være effektive innenfor området vaskulære sykdommer, slik som restenoser, med den samtidige gunstige effekt av øket toleranse. It was chosen to administer flavopiridol orally at a concentration of 5 mg/kg, half the dose that inhibits tumor growth in a nude mouse xenograft model 21. It is noteworthy that flavopiridol concentrations of 75 nmol/1 result in almost complete inhibition of SMC proliferation in the present studies, while mean serum concentrations of 425 nmol/1 were obtained at doses below the toxic limit in phase 1 human studies on refractory carcinoma<17>. Present results suggest that much lower doses of cell cycle inhibitors than those used in neoplasias can be effective in the area of vascular diseases, such as restenoses, with the simultaneous beneficial effect of increased tolerance.
Mens det er vist at flavopiridol induserer vekststans uten å påvirke levedyktigheten til HASMC i kultur, og er vist redusert neointimaldannelse etter flavopiridolbehandling in vivo, kan man ikke være sikker på at cellesyklusarrestasjon er den eneste faktor som reduserer neointimaldannelse ved carotislesjoner. Flavopiridol kan indusere vekststans med eller uten å indusere apoptose, avhengig av celletypen som observeres<29>. Interessant er det at flavopiridol inhiberer apoptose i PC12-celler som er endelig differensiert, mens den induserer apoptose i udifferensierte PC12-celler som er prolifererende<28>. Selv om foreliggende in vitro eksperimenter ble utført under betingelser som etterlikner fenotypen til SMC før skade, er det mulig at SMC kan respondere forskjellig på flavopiridol etter skade og kan til og med gjennomgå apoptose. Mens rollen til apoptose ved vaskulære lesjoner er uklar, induserer ekspresjon av Fas-liganden i SMC apoptose og blokkerer neointimaldannelse hos rotter etter ballongskade<37>, hvilket antyder at dersom flavopiridol virkelig induserer apoptose i SMC in vivo, som den gjør i prolifererende PC12-celler, kan dette være et gunstig fenomen innenfor rammen av neointimaldannelse. Andre mekanismer kan også bidra til effekten av flavopiridol på lesjonsdannelse. For eksempel forbedrer antisense-oligodeoksynukleotidmediert cellesyklusinhibisjon endotelfunksjonen i kaninvenetransplantat<38>. Ytterligere studier vil være nødvendige for å studere mekanismene til flavopiridols effekter, andre enn vekstarrestasjon, på SMC in vivo. While flavopiridol has been shown to induce growth arrest without affecting the viability of HASMC in culture, and has been shown to reduce neointimal formation after flavopiridol treatment in vivo, one cannot be sure that cell cycle arrest is the only factor that reduces neointimal formation in carotid lesions. Flavopiridol can induce growth arrest with or without inducing apoptosis, depending on the cell type observed<29>. Interestingly, flavopiridol inhibits apoptosis in terminally differentiated PC12 cells, while it induces apoptosis in undifferentiated PC12 cells that are proliferating<28>. Although the present in vitro experiments were performed under conditions that mimic the phenotype of SMC before injury, it is possible that SMC may respond differently to flavopiridol after injury and may even undergo apoptosis. While the role of apoptosis in vascular lesions is unclear, expression of the Fas ligand in SMC induces apoptosis and blocks neointimal formation in rats after balloon injury<37>, suggesting that if flavopiridol does indeed induce apoptosis in SMC in vivo, as it does in proliferating PC12- cells, this may be a favorable phenomenon within the framework of neointimal formation. Other mechanisms may also contribute to the effect of flavopiridol on lesion formation. For example, antisense oligodeoxynucleotide-mediated cell cycle inhibition improves endothelial function in rabbit vein grafts<38>. Further studies will be necessary to study the mechanisms of flavopiridol's effects, other than growth arrest, on SMC in vivo.
Gitt den demonstrerte rolle til SMC-proliferering ved lesjonsdannelse etter rotte carotisskade<2>'<3>, er det interessant å bemerke at, til tross for den tydelige effekt av flavopiridol in vitro, var dens evne til å inhibere neointimaldannelse, selv om den var signifikant; mer moderat under betingelser ifølge foreliggende in vivo eksperimenter. Flere ulike forklaringer er vurdert for denne observasjon. Det er usannsynlig at akselerert SMC-proliferering foregår etter opphør av flavopiridol, siden forskjeller i proliferativ indeks vedvarer så lenge som 14 dager etter skade (figur 7 og 9). Det er mer sannsynlig at enten andre komponenter ved lesjonsdannelse, slik som SMC-migrering og ekstracellulær matriksproduksjon, fortsatt bidrar til lesjonsdannelse, selv i fravær av betydelig SMC-proliferering, eller at avlevering av flavopiridol en gang daglig er utilstrekkelig for å arrestere proliferasjonen fullstendig i denne modell. Nylige data antyder at den biologiske halveringstid på flavopiridol er så kort som 2,5 timer, hvilket antyder at den sistnevnte hypotese kan være korrekt; flere studier kan gjøre det mulig å identifisere et enda mer effektivt doseregime<39>. Given the demonstrated role of SMC proliferation in lesion formation after rat carotid injury<2>'<3>, it is interesting to note that, despite the clear effect of flavopiridol in vitro, its ability to inhibit neointimal formation, although the was significant; more moderately under conditions according to the present in vivo experiments. Several different explanations have been considered for this observation. It is unlikely that accelerated SMC proliferation occurs after cessation of flavopiridol, since differences in proliferative index persist as long as 14 days after injury (Figures 7 and 9). It is more likely that either other components of lesion formation, such as SMC migration and extracellular matrix production, still contribute to lesion formation, even in the absence of significant SMC proliferation, or that once-daily delivery of flavopiridol is insufficient to completely arrest proliferation in this model. Recent data suggest that the biological half-life of flavopiridol is as short as 2.5 hours, suggesting that the latter hypothesis may be correct; more studies may make it possible to identify an even more effective dose regimen<39>.
Foreliggende resultater antyder at flavopiridol kan inhibere SMC-proliferering og derfor neointimaldannelse i en vel akseptert modell for vaskulær sykdom i smådyr. Det må poengteres at relevansen av inhibisjon av SMC-proliferering er kontroversiell ved humane, vaskulære lesjoner, og kan være forskjellig avhengig av egenskapene til lesjonen og tidspunktet for når observasjoner på proliferasjon er utført. Den proliferative indeks til SMC i humane aterektomiprøver er bemerkelsesverdig lav<40>, selv om disse prøver ikke nødvendigvis gjenspeiler proliferative endringer ved tidligere, mer kritiske, stadier av lesjonsutviklingen. I tillegg kan remodellering av arterier uavhengig av neointimal vekst utgjøre en betydelig andel av den luminale obstruksjon etter angioplasti hos menneske<41>.1 motsetning er mitotisk aktivitetsindeks i SMC langt høyere (25 % PCNA-positive) i aterektomiprøver fra menneskelige lesjoner med "in-stent" restenoser, hvilket er konsistent med den etablerte rolle til SMC-hyperplasi, men ikke remodellering i denne prosess<4>. Ettersom plassering av stenter og kliniske problemer ved restenoser med stenter øker, vil det være behov for en effektiv metode for å stanse SMC-hyperplasi og neointimaldannelse. Siden flavopiridol er et potent, oralt tilgjengelig medikament med spesifikk Cdk-inhibitorisk aktivitet, og siden sikre doser av flavopiridol er kjent for menneske, kan flavopiridol vurderes som en farmasøytisk kandidat for forebyggelse av restenoser ved stenter hos mennesker. Present results suggest that flavopiridol can inhibit SMC proliferation and therefore neointimal formation in a well-accepted model of vascular disease in small animals. It must be pointed out that the relevance of inhibition of SMC proliferation is controversial in human vascular lesions, and may differ depending on the characteristics of the lesion and the time at which proliferation observations are made. The proliferative index of SMC in human atherectomy specimens is remarkably low<40>, although these specimens do not necessarily reflect proliferative changes at earlier, more critical, stages of lesion development. In addition, arterial remodeling independent of neointimal growth may account for a significant proportion of the luminal obstruction after angioplasty in humans<41>.1 In contrast, the mitotic activity index in SMC is far higher (25% PCNA-positive) in atherectomy specimens from human lesions with "in -stent" restenoses, which is consistent with the established role of SMC hyperplasia but not remodeling in this process<4>. As the placement of stents and the clinical problems of restenosis with stents increase, there will be a need for an effective method to arrest SMC hyperplasia and neointimal formation. Since flavopiridol is a potent, orally available drug with specific Cdk inhibitory activity, and since safe doses of flavopiridol are known for humans, flavopiridol can be considered as a pharmaceutical candidate for the prevention of stent restenosis in humans.
Fremgangsmåter og resultater: Ved anvendelse av dyrkede, humane aorta muskelceller ble det funnet at flavopiridol i konsentrasjoner så lave som 75 nmol/1 resulterte i nesten fullstendig inhibisjon av basisk fibroblastvekstfaktor og trombinindusert proliferering. Ved denne dose inhiberte flavopiridol syklinavhengig kinaseaktivitet, målt ved histon Hi-fosforylering, men hadde ingen effekt på MAP-kinaseaktivitet. Induksjon av det cellesyklusrelaterte protein syklin Di, prolifererende cellekjerneantigen og fosforylert retinablastomprotein ble også blokkert av flavopiridol. Flavopiridol hadde ingen effekt på cellelevedyktighet. For å teste om flavopiridol hadde en tilsvarende aktivitet in vivo når den ble administrert oralt, ble neointimaldannelse i rotte carotisarterier etter ballongskade undersøkt. Flavopiridol (5 mg/kg) administrert ved sondeforing reduserte det neointimale området med 35 % og 39 %, henholdsvis 7 og 14 dager etter skade. Methods and Results: Using cultured human aortic muscle cells, it was found that flavopiridol at concentrations as low as 75 nmol/1 resulted in almost complete inhibition of basic fibroblast growth factor and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone Hi phosphorylation, but had no effect on MAP kinase activity. Induction of the cell cycle-related protein cyclin Di, proliferating cell nuclear antigen and phosphorylated retinablastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cell viability. To test whether flavopiridol had a similar activity in vivo when administered orally, neointimal formation in rat carotid arteries after balloon injury was investigated. Flavopiridol (5 mg/kg) administered by gavage reduced the neointimal area by 35% and 39%, 7 and 14 days after injury, respectively.
Konklusjon: Flavopiridol inhiberer SMC-vekst in vitro og in vivo. Dens orale tilgjengelighet og selektivitet for syklinavhengige kinaser, gjør den til et potensielt terapeutisk verktøy ved behandling av SMC-rike vaskulære lesjoner. Conclusion: Flavopiridol inhibits SMC growth in vitro and in vivo. Its oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.
REFERANSER REFERENCES
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. 1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s.
Nature 1993; 362:801-9. 2. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab. Invest. 1983; 49:327-333. 3. Clowes A, Schwartz S. Significance of quiescent smooth muscle migration in the injuried rat carotid artery. Circ. Res. 1985; 56:139-145. 4. Kearney M, Pieczek A, Haley L, Losordo DW, Andres V, Schainfeld R, Rosenfield K, Isner JM. Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 1997; 95: 1998 -2002. 5. Mintz GS, Hoffmann R, Mehran R, Pichard AD, Kent KM, Satler LF, Popma JJ, Leon MB. In-stent restenosis: the Washington Hospital Center experience. Am. J. Nature 1993; 362:801-9. 2. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab. Invest. 1983; 49:327-333. 3. Clowes A, Schwartz S. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ. Res. 1985; 56:139-145. 4. Kearney M, Pieczek A, Haley L, Losordo DW, Andres V, Schainfeld R, Rosenfield K, Isner JM. Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 1997; 95: 1998 -2002. 5. Mintz GS, Hoffmann R, Mehran R, Pichard AD, Kent KM, Satler LF, Popma JJ, Leon MB. In-stent restenosis: the Washington Hospital Center experience. Am. J.
Cardiol. 1998; 81:7E-13E. Cardiol. 1998; 81:7E-13E.
6. Califf RM. Restenosis: the cost to society. Am. Heart J. 1995; 130:680-684. 6. Califf RM. Restenosis: the cost to society. Am. Heart J. 1995; 130:680-684.
7. Sherr CJ. Mammalian Gi cyclins. Cell 1993; 73:1059-1065. 7. Sherr CJ. Mammalian Gi cyclins. Cell 1993; 73:1059-1065.
8. Hunter T. Braking the cycle. Cell 1993; 75:839-841. 8. Hunter T. Braking the cycle. Cell 1993; 75:839-841.
9. Chen D, Krasinski D, Chen D, Sylvester A, Chen J, Nisen PD, Andres V. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27<Kn>1>, an inhibitor of neointima 9. Chen D, Krasinski D, Chen D, Sylvester A, Chen J, Nisen PD, Andres V. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27<Kn>1>, an inhibitor of neointima
formation in the rat carotid artery. J. Clin. Invest. 1997; 99:2334-2341. formation in the rat carotid artery. J. Clin. Invest. 1997; 99:2334-2341.
10. Chang MW, Barr E, Lu MM, Barton K,Leiden JM. Adenovirus-mediated overexpression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery 10. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated overexpression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery
model of balloon angioplasty. J. Clin. Invest. 1995; 96:2260-2268. model of balloon angioplasty. J. Clin. Invest. 1995; 96:2260-2268.
11. Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular proliferative disorders with a constitutively 11. Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular proliferative disorders with a constitutive
active form of the retinoblastoma gene product. Science 1995; 267:518-522. active form of the retinoblastoma gene product. Science 1995; 267:518-522.
12. Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. A Gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proe. Nati. 12. Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Pro. Nati.
Acad. Sei. USA 1995; 92:5855-5859. 13. Losiewitz MD, Carlson BA, Kaur G, Sausville EA, Worland PJ. Potent inhibition of Cdc2 kinase activity by the flavanoid, L86-8275. Biochem. Biophys. Res. Commun. Acad. Pollock. United States 1995; 92:5855-5859. 13. Losiewitz MD, Carlson BA, Kaur G, Sausville EA, Worland PJ. Potent inhibition of Cdc2 kinase activity by the flavanoid, L86-8275. Biochem. Biophys. Res. Commun.
1994; 201:589-595. 1994; 201:589-595.
14. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces Gi arrest with inhibition of cyclin-dependent kinase (CDK) 2 og cdk4 in 14. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces Gi arrest with inhibition of cyclin-dependent kinase (CDK) 2 and cdk4 in
human breast carcinoma cells. Canc. Res. human breast carcinoma cells. Canc. Res.
15. de Azevedo WF, Mueller-Dieckmann H-J, Schuze-Gahmen U, Worland PJ, Sausville E, Kim S-H. Structual basis for specificity and potency of a flavanoid inhibitor of human Cdk2, a cell cycle kinase. Proe. Nati. Acad. Sei. USA 1996; 15. de Azevedo WF, Mueller-Dieckmann H-J, Schuze-Gahmen U, Worland PJ, Sausville E, Kim S-H. Structural basis for specificity and potency of a flavanoid inhibitor of human Cdk2, a cell cycle kinase. Pro. Nati. Acad. Pollock. United States 1996;
93:2735-2740. 93:2735-2740.
16. Kaur G, Stetler-Stevenson M, Sebers S, Worland P, Sedlacek H, Myers C, Czech J, Naik R, Sausville E. Growth inhibition with reversible cell cycle arrest of carcinoma 16. Kaur G, Stetler-Stevenson M, Sebers S, Worland P, Sedlacek H, Myers C, Czech J, Naik R, Sausville E. Growth inhibition with reversible cell cycle arrest of carcinoma
cells by flavone L86-8275. J. Nati. Canc. Inst. 1992; 84:1736-1740. cells by flavone L86-8275. J. Nati. Canc. Institute 1992; 84:1736-1740.
17. Senderowicz AM, Headlee D, Stinson S, Lush RM, Figg WD, Pluda J, Sausville Ea. A Phase I trial of flavopiridol (FLA), a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasm. Proe. Am. Soc. Clin. Oncol. 1997; 16:226a. 17. Senderowicz AM, Headlee D, Stinson S, Lush RM, Figg WD, Pluda J, Sausville Ea. A Phase I trial of flavopiridol (FLA), a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasm. Pro. Am. Soc. Clin. Oncol. 1997; 16:226a.
Abstract. Abstract.
18. Ruef J, Hu ZY, Yin L-Y, Wu Y, Hanson sr, Kelly AB, Harker LA, Rao Gn, Runge MS, Patterson C. Induction of vascular endothelial growth factor in ballooninjured 18. Ruef J, Hu ZY, Yin L-Y, Wu Y, Hanson sr, Kelly AB, Harker LA, Rao Gn, Runge MS, Patterson C. Induction of vascular endothelial growth factor in ballooninjured
baboon arteries. Circ. Res. 1997; 81:24-33. baboon arteries. Circ. Res. 1997; 81:24-33.
19. Ruef J, Rao Gn, Li F, Bode C, Patterson C, Bhatnagar A, Runge MS. Induction of rat aortic smooth muscle cell growth by the lipid peroxidation product 4-hydroxy-2-noneal. Circulation 1998; 97:1071-1078. 20. Swank Ra, T'ng Jp, Guo XW, Valdez J, Bradbury EM, Gurley LR. Four distinct cyclin-dependent kinases phosphorylate histone Hi at all of its growth related 19. Ruef J, Rao Gn, Li F, Bode C, Patterson C, Bhatnagar A, Runge MS. Induction of rat aortic smooth muscle cell growth by the lipid peroxidation product 4-hydroxy-2-noneal. Circulation 1998; 97:1071-1078. 20. Swank Ra, T'ng Jp, Guo XW, Valdez J, Bradbury EM, Gurley LR. Four distinct cyclin-dependent kinases phosphorylate histone Hi at all of its growth related
phosphorylation sites. Biochemistry 1997; 36:13761-13768. phosphorylation sites. Biochemistry 1997; 36:13761-13768.
21. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 regulates novel cyclins during the Gi phase of the cell cycle. Cell 1991; 65:701-713. 22. Bravo R. Synthesis of the nuclear protein (PCNA) and its relationships with DNA replication. Exp. Cell Res. 1986; 163:287-293. 23. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato JY, Livingston DM. Functional interaction of the retinoblastoma protein with mammalian D-type cyclins. Cell 1993; 7:487-497. 24. Hunter T. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell 1995; 80:225-236. 25. Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBOJ. 1991; 10:885-892. 26. Konig A, Schwartz GK, Mohammad RM, Al-katib A, Gabrilove JL. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 1997; 90:4307-4312. 27. Drees M, Dengler WA, Roth T, Labonte H, Mayo J, Malspeis L, Grever M, Sausville EA, Fieberg HH. Flavopiridol (L86-8275): Selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. Clin. Cancer Res. 1997; 21. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 regulates novel cyclins during the Gi phase of the cell cycle. Cell 1991; 65:701-713. 22. Bravo R. Synthesis of the nuclear protein (PCNA) and its relationships with DNA replication. Exp. Cell Res. 1986; 163:287-293. 23. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato JY, Livingston DM. Functional interaction of the retinoblastoma protein with mammalian D-type cyclins. Cell 1993; 7:487-497. 24. Hunter T. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signalling. Cell 1995; 80:225-236. 25. Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBOY. 1991; 10:885-892. 26. Konig A, Schwartz GK, Mohammad RM, Al-katib A, Gabrilove JL. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 1997; 90:4307-4312. 27. Drees M, Dengler WA, Roth T, Labonte H, Mayo J, Malspeis L, Grever M, Sausville EA, Fieberg HH. Flavopiridol (L86-8275): Selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. Clin. Cancer Res. 1997;
3:273-279. 3:273-279.
28. Park DS, Rarinelli SE, Greene LA. Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC 12 cells and sympathetic 28. Park DS, Rarinelli SE, Greene LA. Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC 12 cells and sympathetic
neurons. J. Biol. Chem. 1996; 271:8161-8169. neurons. J. Biol. Chem. 1996; 271:8161-8169.
29. Parker BW, Kaur G, Nieves-Niera W, Taimi M, Kohlhagen G, Shimizu T, Losiewicz MD, Pommier Y, Sausville EA, Senderowicz AM. Early induction of apoptosis in hematopoetic cell lines after exposure to flavopiridol. Blood 1998; 29. Parker BW, Kaur G, Nieves-Niera W, Taimi M, Kohlhagen G, Shimizu T, Losiewicz MD, Pommier Y, Sausville EA, Senderowicz AM. Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 1998;
91:458-465. 91:458-465.
30. Geng YJ, Wu Q, Muszynski M, Hansson GK, Libby P. Apoptosis of vascular smooth muscle cells induced by in vitro stimaltion with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta. Arterioscler. Thromb. Biol. 1996; 16:19-27. 31. Schwartz SM, deBlois D, 0'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ. Res. 1995; 77:445-465. 32. Wei GL, Krasinkski K, Kearney M, Isner JM, Walsh K, Andres V. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. 30. Geng YJ, Wu Q, Muszynski M, Hansson GK, Libby P. Apoptosis of vascular smooth muscle cells induced by in vitro stimulation with interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 beta. Arteriosclerosis. Thromb. Biol. 1996; 16:19-27. 31. Schwartz SM, deBlois D, 0'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ. Res. 1995; 77:445-465. 32. Wei GL, Krasinkski K, Kearney M, Isner JM, Walsh K, Andres V. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty.
Circ. Res. 1997; 80:418-426. 33. de Young MB, Dichek DA. Gene therapy for restenosis. Circ. Res. 1997; 82:306-313. 34. Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, Norman TC, Rosete J, Rowe M, Schow SR, Schultz TG, Wang X, Wick MM, Shiffman D. CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal formation. J. Circ. Res. 1997; 80:418-426. 33. de Young MB, Dichek DA. Gene therapy for restenosis. Circ. Res. 1997; 82:306-313. 34. Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, Norman TC, Rosete J, Rowe M, Schow SR, Schultz TG, Wang X, Wick MM, Shiffman D. CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal formation. J.
Biol. Chem. 1997; 272:299207-29911. Biol. Chem. 1997; 272:299207-29911.
35. Sirois MG, Simons M, Edelman ER. Antisense olignucleotide inhibition of PDGFR-b receptor subunit expression directs suppression of intimal thickening. Circulation 35. Sirois MG, Simons M, Edelman ER. Antisense oligonucleotide inhibition of PDGFR-b receptor subunit expression directs suppression of intimal thickening. Circulation
1997; 95:669-676. 36. Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. 1997; 95:669-676. 36. Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury.
Nature Med. 1996;2:293-298. Nature Med. 1996;2:293-298.
37. Sata M, Perlman H, Muruve DA, Silver M, Ikebe M, Libermann TA, Oettgen P, Walsh K. Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response. Proe. Nati. Acad. Sei. USA 37. Sata M, Perlman H, Muruve DA, Silver M, Ikebe M, Libermann TA, Oettgen P, Walsh K. Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response. Pro. Nati. Acad. Pollock. USA
1998; 95:1213-1217. 1998; 95:1213-1217.
38. Mann MJ, Gibbons GH, Tsao PS, von der Leyen HE, Cooke JP, Buitrago R, Kernoff R, Dzau VJ. Cell cycle inhibition preserves endothelial function in 38. Mann MJ, Gibbons GH, Tsao PS, von der Leyen HE, Cooke JP, Buitrago R, Kernoff R, Dzau VJ. Cell cycle inhibition preserves endothelial function in
genetically engineered rabbit vein grafts. J. Clin. Invest. 1997; 99:1295-1301. genetically engineered rabbit vein grafts. J. Clin. Invest. 1997; 99:1295-1301.
39. Arguello F, Alexander M, Sterry JA, Tudor G, Smith EM, Kalavar NT, Greene JF, Koss W, Morgan CD, Stinson SF, Siforf TJ, Alvord WG, Klabansky RL, Sausville EA: Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against human 39. Arguello F, Alexander M, Sterry JA, Tudor G, Smith EM, Kalavar NT, Greene JF, Koss W, Morgan CD, Stinson SF, Siforf TJ, Alvord WG, Klabansky RL, Sausville EA: Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against humans
leukemia and lymphoma xenografts. Blood 1998; 91:2482-2490. leukemia and lymphoma xenografts. Blood 1998; 91:2482-2490.
40. 0'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM. Proliferation in primary and restenotic coronary atherectomy tissue: implications for anti-proliferative therapy. Circ. Res. 40. 0'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM. Proliferation in primary and restenotic coronary atherectomy tissue: implications for anti-proliferative therapy. Circ. Res.
1993;73:223-231. 41. Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LR, Wong SC, Hong MD, Kovach JA, Leon MB. Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation 1996; 94:35-43. 1993;73:223-231. 41. Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LR, Wong SC, Hong MD, Kovach JA, Leon MB. Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation 1996; 94:35-43.
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Families Citing this family (58)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB0102480D0 (en) * | 2001-01-31 | 2001-03-14 | Cyclacel Ltd | Marker |
DK1511734T3 (en) * | 2002-04-17 | 2010-08-02 | Cytokinetics Inc | Compounds, Preparations and Methods |
US7425618B2 (en) | 2002-06-14 | 2008-09-16 | Medimmune, Inc. | Stabilized anti-respiratory syncytial virus (RSV) antibody formulations |
US7132100B2 (en) | 2002-06-14 | 2006-11-07 | Medimmune, Inc. | Stabilized liquid anti-RSV antibody formulations |
US20040082613A1 (en) * | 2002-06-28 | 2004-04-29 | Schneider Michael D. | Modulators of Cdk9 as a therapeutic target in cardiac hypertrophy |
US20040106647A1 (en) * | 2002-06-28 | 2004-06-03 | Schneider Michael D. | Modulators of Cdk9 as a therapeutic target in cardiac hypertrophy |
AU2003273299B2 (en) * | 2002-09-05 | 2010-04-01 | Medimmune, Llc | Methods of preventing or treating cell malignancies by administering CD2 antagonists |
US7563810B2 (en) | 2002-11-06 | 2009-07-21 | Celgene Corporation | Methods of using 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione for the treatment and management of myeloproliferative diseases |
US8034831B2 (en) | 2002-11-06 | 2011-10-11 | Celgene Corporation | Methods for the treatment and management of myeloproliferative diseases using 4-(amino)-2-(2,6-Dioxo(3-piperidyl)-isoindoline-1,3-dione in combination with other therapies |
WO2004091510A2 (en) | 2003-04-11 | 2004-10-28 | Medimmune, Inc. | Recombinant il-9 antibodies and uses thereof |
ES2458636T3 (en) | 2003-08-18 | 2014-05-06 | Medimmune, Llc | Humanization of antibodies |
US20060228350A1 (en) * | 2003-08-18 | 2006-10-12 | Medimmune, Inc. | Framework-shuffling of antibodies |
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CA2613512A1 (en) | 2005-06-23 | 2007-01-04 | Medimmune, Inc. | Antibody formulations having optimized aggregation and fragmentation profiles |
WO2007010342A1 (en) * | 2005-07-15 | 2007-01-25 | Sahajanand Biotech Private Limited | Implantable medical devices comprising a flavonoid or derivative thereof for prevention of restenosis |
JP2008255008A (en) * | 2005-07-19 | 2008-10-23 | Tokyo Medical & Dental Univ | Synoviocyte proliferation inhibitor |
AU2006275514B2 (en) * | 2005-07-29 | 2012-04-05 | Resverlogix Corp. | Pharmaceutical compositions for the prevention and treatment of complex diseases and their delivery by insertable medical devices |
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EP2346837B8 (en) | 2008-06-26 | 2015-04-15 | Resverlogix Corporation | Methods of preparing quinazolinone derivatives |
EP2382194B1 (en) | 2009-01-08 | 2014-03-12 | Resverlogix Corp. | Compounds for the prevention and treatment of cardiovascular disease |
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WO2012103165A2 (en) | 2011-01-26 | 2012-08-02 | Kolltan Pharmaceuticals, Inc. | Anti-kit antibodies and uses thereof |
PL2773354T3 (en) | 2011-11-01 | 2019-12-31 | Resverlogix Corp. | Oral immediate release formulations for substituted quinazolinones |
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WO2014080290A2 (en) | 2012-11-21 | 2014-05-30 | Rvx Therapeutics Inc. | Cyclic amines as bromodomain inhibitors |
JP2016507496A (en) | 2012-12-21 | 2016-03-10 | ゼニス・エピジェネティクス・コーポレイションZenith Epigenetics Corp. | Novel heterocyclic compounds as bromodomain inhibitors |
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CA3006769A1 (en) | 2015-12-02 | 2017-06-08 | Stcube & Co., Inc. | Antibodies and molecules that immunospecifically bind to btn1a1 and the therapeutic uses thereof |
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CA3034875C (en) | 2016-08-23 | 2024-05-28 | Eisai R&D Management Co., Ltd. | Combination therapies for the treatment of hepatocellular carcinoma |
WO2018083248A1 (en) | 2016-11-03 | 2018-05-11 | Kymab Limited | Antibodies, combinations comprising antibodies, biomarkers, uses & methods |
WO2018094275A1 (en) | 2016-11-18 | 2018-05-24 | Tolero Pharmaceuticals, Inc. | Alvocidib prodrugs and their use as protein kinase inhibitors |
MA47776A (en) | 2017-03-16 | 2020-01-22 | Eisai R&D Man Co Ltd | POLYTHERAPIES FOR THE TREATMENT OF BREAST CANCER |
KR20200015602A (en) | 2017-05-31 | 2020-02-12 | 주식회사 에스티큐브앤컴퍼니 | Antibodies and molecules immunospecifically binding to BTN1A1 and therapeutic uses thereof |
US20200131266A1 (en) | 2017-05-31 | 2020-04-30 | Stcube & Co., Inc. | Methods of treating cancer using antibodies and molecules that immunospecifically bind to btn1a1 |
EP3635007A1 (en) | 2017-06-06 | 2020-04-15 | STCube & Co., Inc. | Methods of treating cancer using antibodies and molecules that bind to btn1a1 or btn1a1-ligands |
JP7196160B2 (en) | 2017-09-12 | 2022-12-26 | スミトモ ファーマ オンコロジー, インコーポレイテッド | Treatment Regimens for Cancers Insensitive to BCL-2 Inhibitors Using the MCL-1 Inhibitor Albocidib |
CN111630069B (en) | 2017-10-13 | 2024-05-31 | 勃林格殷格翰国际有限公司 | Human antibodies to Thomsen-non-velle (Tn) antigen |
AU2019306165A1 (en) | 2018-07-20 | 2021-02-25 | Pierre Fabre Medicament | Receptor for vista |
AU2019391097A1 (en) | 2018-12-04 | 2021-05-20 | Sumitomo Pharma Oncology, Inc. | CDK9 inhibitors and polymorphs thereof for use as agents for treatment of cancer |
US11793802B2 (en) | 2019-03-20 | 2023-10-24 | Sumitomo Pharma Oncology, Inc. | Treatment of acute myeloid leukemia (AML) with venetoclax failure |
CN113559244B (en) * | 2021-08-02 | 2023-12-26 | 华中科技大学同济医学院附属协和医院 | New application of CTRP13 fat factor |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IN164232B (en) * | 1986-04-11 | 1989-02-04 | Hoechst India | |
US5284856A (en) * | 1988-10-28 | 1994-02-08 | Hoechst Aktiengesellschaft | Oncogene-encoded kinases inhibition using 4-H-1-benzopyran-4-one derivatives |
US5733920A (en) | 1995-10-31 | 1998-03-31 | Mitotix, Inc. | Inhibitors of cyclin dependent kinases |
US5849733A (en) | 1996-05-10 | 1998-12-15 | Bristol-Myers Squibb Co. | 2-thio or 2-oxo flavopiridol analogs |
US5908934A (en) | 1996-09-26 | 1999-06-01 | Bristol-Myers Squibb Company | Process for the preparation of chiral ketone intermediates useful for the preparation of flavopiridol and analogs |
JP2001509805A (en) | 1997-02-05 | 2001-07-24 | ワーナー−ランバート・コンパニー | Pyrido [2,3-D] pyrimidine and 4-aminopyrimidine as cell growth inhibitors |
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- 2001-07-27 BG BG105751A patent/BG65151B1/en unknown
- 2001-08-23 CR CR6448A patent/CR6448A/en not_active Application Discontinuation
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2002
- 2002-05-31 HK HK02104062.3A patent/HK1042445B/en not_active IP Right Cessation
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